This invention relates to microcontact printing (i.e., microstamping) of biomolecules and other ligands onto polymer surfaces.
Micropatterning of biomolecules on surfaces has a number of applications, including the modulation of cell-substrate interactions in biomaterials and tissue engineering and the fabrication of multi-analyte biosensors and genomic arrays. See Blawas, A. S. et al, Biomaterials (1998), 19, 595; Mrksich, M. and Whitesides, G. M., TIBTECH (1995) 13, 228. Microcontact printing (also referred to herein as xe2x80x9cxcexcCPxe2x80x9d) methods are attractive for micropatterning of biomolecules, because of their simplicity and ease of use. See Kumar, A. et al., Acc. Chem. Res. 1995, 28, 219; Xia, Y. et al., Angew. Chem. Int. Ed. Engl. (1998), 37, 550. To date, however, methods of microstamping have generally been limited to the production of patterns on self assembling monolayers (SAMs), which in turn are bound to gold or silicon surfaces. For example, Whitesides and coworkers have used reactive xcexcCP to pattern biological ligands onto reactive SAMs on gold. J. Lahiri, et al., Langmuir 1999, 15, 2055. Bernard et al. have similarly used xcexcCP to pattern different proteins onto SAMs on gold by physical adsorption. See Bernard, A.; et al., Langmuir 1998, 14, 2225.
U.S. Pat. No. 5,512,131 to Kumar et al. describes a method of patterning a surface in which an elastomeric stamp with a stamping surface is coated with a self-assembled monolayer-forming species having a functional group selected to bind to surface. The stamp is then placed against the surface to leave a self-assembled monolayer of the species originally coated onto the stamp. The description of the invention is, however, limited to the use of self-assembling monolayers. While SAMs are commonly used, the limitation of being required to use them is disadvantageous in that SAMs generally bind only to certain materials such as metals (usually gold), silicon dioxide, gallium arsenide, glass, and the like. The patent fails to provide any example of a non-SAM species being used to bind directly to a surface, nor does the patent recite any examples of microstamping onto a material other than gold.
While SAMs on gold are generally used for micropatterning, they have limited utility as biomaterials. In contrast, polymers are widely used as biomaterials. (Zdrahala, R. J., J. Biomater. Appl. (1996) 10, 309). Most previous studies on micropatterning on polymers have utilized photolithography. (Mooney, J. F. et al., Proc. Natl. Acad. Sci. (USA) 1996, 93, 12287. Wybourne, M. N. et al., Nanotechnology 1996, 7, 302. Hengsakul, M et al., Bioconj. Chem. 1996, 7, 249. Schwarz, A. et al., Langmuir 1998, 14, 552; Dewez, J.-L. et al., Biomaterials 1998, 19). Alternative methods have also been demonstrated by Ghosh and Crooks, who patterned hyperbranched poly(acrylic acid) on oxidized poly(ethylene) using reactive xcexcCP. (Ghosh, P.; Crooks, R. M. J. Am. Chem. Soc., 1999,121, 8395).
The micropatterning of biological molecules onto surfaces is an important objective because such patterning enables, for example, control of cell-substrate interactions. (Chen, C. S.; et al., Science 1997, 276, 1425. Mrksich, M. et al., Exp. Cell Res. 1997, 235, 305. Chen, C. S; et al., Whitesides, G. M. et al., Biotechnol. Prog. 1998, 14, 356). In the last decade, biomolecules have been immobilized onto the surface of different polymers in order to modulate their interaction with cells. (Shakesheff, K. et al., J. Biomater. Sci., Polym. Ed. 1998, 9, 507; Cima, L. G., J. Cell. Biochem. 1994, 56, 155; Massia, S. P. et al., J. Biomed. Mater. Res. 1991, 25, 223; Brandley, B. K.; et al., Anal. Biochem. 1988, 172, 270. Massia, S. P. et al., Anal. Biochem. 1990, 187, 292). More recent studies have focused on patterning polymer surfaces with biological ligands. Mooney, J. F.; Hunt, et al., Proc. Natl. Acad. Sci. (USA) 1996, 93, 12287. Wybourne, M. N. et al., Nanotechnology 1996, 7, 302; Hengsakul, M. et al., Bioconj. Chem. 1996, 7, 249; Schwarz, A.; et al., Langmuir 1998, 14, 5526; Dewez, J.-L. et al., P. G. Biomaterials 1998, 19).
Despite the foregoing, current attempts to micropattern biological ligands onto polymer surfaces are severely limited. Most xcexcCP methods are done and indeed are required to be performed on gold or similar metal surfaces. Typically, a SAM-molecule is stamped onto a gold surface to create a patterned SAM layer on the gold surface. See Kumar, A. et al., supra. In a modification of this basic method, Lahiri et al., supra, have developed a method in which a homogeneous SAM is formed on gold by incubating the gold surface in a solution of the SAM-forming molecules. Next, a stamp is used to transfer a non-SAM reactive molecule to the SAM/gold surface. The reactive molecule reacts with a with a reactive molecule in the SAM to form a pattern of the reactive molecule on the SAM/gold surface. These methods are limiting because they are restricted to the use of gold or other SAM forming surfaces, and require the use of SAM-forming molecules. These approaches are not applicable to polymer surfaces because SAMs do not generally form on polymers. In yet another alternative approach (Barnard et al., supra), a stamp xe2x80x9cinkedxe2x80x9d with protein is used to stamp a pattern of the protein onto a polymer. A significant limitation of this method is that the protein is not bound to the polymer surface via a stable, covalent linkage or bond. Rather, the protein is attached to the polymer surface by physical adsorption. This approach is limiting because many molecules of interest cannot be stably bound to polymer surfaces by non-specific physical adsorption, and the patterned molecule is easily removed from the polymer surface by water, buffers, biological fluids and the like.
Thus, the successful patterning of biological ligands directly onto polymer surfaces using reactive microstamping techniques (i.e., in which reactions between the ligands and the polymer surfaces occur to create a stable covalent bond between the two) has heretofore remained elusive. Accordingly, a need exists for a reliable method of microstamping biological and other ligands directly and covalently onto polymer surfaces.
The present inventor has discovered a novel methodology of reactive xcexcCP microstamping that overcomes many of the shortcomings presented by the present available methods. This methodology enables biological ligands and proteins to be directly patterned on polymers with a spatial resolution of at least 5 xcexcm and good reproducibility. In addition to providing a desirable level of resolution, the methods of the present invention also provide spatial control of ligand presentation on the surface of commonly used polymeric biomaterials.
Accordingly, a first aspect of the present invention is a method of microstamping a polymer surface with a ligand, in which a functionalized polymer surface having a reactive moiety thereon is contacted with a stamp adsorbed onto its surface at least one ligand comprising a second reactive moiety, wherein the second reactive moiety of the ligand and the first reactive moiety of the polymer surface form a covalent bond. After the covalent bond is formed, the stamp is separated from the functionalized polymer surface, thereby leaving the ligand covalently bound to the functionalized polymer surface.
This method results in spatially-resolved transfer and coupling of the ligand to the reactive surface of the polymer. In a preferred embodiment of the invention, the ligand is a biological ligand. However, other ligands, including synthetic polymers, may also be used in the methods of the present invention.
An additional aspect of the invention is a device comprising at least one microstamped polymer surface, wherein the polymer surface is covalently bound to at least one ligand. Methods of forming such devices, such as tissue culture plates, are also an aspect of the invention.
The foregoing and other aspects of the present invention are explained in detail in the specification set forth below.